Development of a novel micro biosensor for in vivo monitoring of glutamate release in the brain

L- Glutamate is the main excitatory neurotransmitter in the central nervous system and hyperglutamatergic signaling is implicated in neurological and neurodegenerative diseases. Monitoring glutamate with a glutamate oxidase-based amperometric biosensor offers advantages such as high spatial and high temporal resolution. However, commercially-available glutamate biosensors are expensive and larger in size. Here, we report the development of 50 gm diameter biosensor for real-time monitoring of L-glutamate in vivo. A polymer, poly-o-phenylenediamine (PPD) layer was electropolymerized onto a 50 mu m Pt wire to act as a permselective membrane. Then, glutamate oxidase entrapped in a biocompatible chitosan matrix was cast onto the microelectrode surface. Finally, ascorbate oxidase was coated to eliminate interferences from high levels of extracellular ascorbic acid present in brain tissue. L-glutamate measurements were performed amperometrically at an applied potential of 0.6 V vs Ag/AgCl. The biosensor exhibited a linear range from 5 to 150 mu M, with a high sensitivity of 0.097 +/- 0.001 nA/mu M and one-week storage stability. The biosensor also showed a rapid steady state response to L-glutamate within 2 s, with a limit of detection of 0.044 mu M. The biosensor was used successfully to detect stimulated glutamate in the subthalamic nucleus in brain slices and in vivo. Thus, this biosensor is appropriate for future neuroscience applications.